Among neutralizing antibodies (Nabs) directed against the HIV-1 envelope gp120/gp41 glycoprotein, 2F5 and 4E10 stand out for their potency and broadly neutralizing activity. Both antibodies are directed against the membrane proximal external region (MPER) of the gp41 stalk. Unfortunately, 2F5 and 4E10-like anti-MPER Nabs are rarely elicited in patients [3;70]. Both of these Nabs differ from the norm in that each of their antigen binding sites contain a long, hydrophobic H chain complementarity determining region 3 (CDR-H3). In previous studies we have shown that immunoglobulins with hydrophobic CDR-H3s are normally culled from the conventional B cell repertoire. We thus propose that 2F5- and 4E10-like Nabs are difficult to elicit because as a result of this normal process the frequency of this type of antigen binding site in healthy individuals tends to be very low. We will test the hypothesis that forced enrichment for use of long, hydrophobic CDR-H3 intervals will promote the production of Nabs directed against the HIV-1 MPER. We also propose to test the complementary hypothesis that the use of these long, hydrophobic CDR-H3 intervals in anti-MPER Nabs may require release from normal tolerance mechanisms. The application is responsive to the RFA in that its focus is basic immunology research on B cell and antibody regulation in the context of HIV-1 vaccine development. Our proposed studies target a critical conserved epitope on the HIV-1 envelope glycoprotein gp120/gp41. They focus on elucidating basic immune mechanisms by which an effective and robust neutralizing antibody response can be elicited in uninfected individuals. They address the issue of tolerance and autoimmunity in eliciting Nabs. And, our studies foster collaboration between two established basic B cell immunologists, Drs. Schroeder and Kearney;and two experts in HIV, Drs. Haynes and Shaw. We have created a strain of mouse, ?D-D[unreadable]FS, where we force use of hydrophobic CDR-H3s by committing B cells to the use of a hydrophobic DH reading frame, valine-enriched DFL16.1 RF2. DFL16.1 is also two to four codons longer than the other mouse DH sequences. Thus, the ?D-D[unreadable]FS mouse creates a polyclonal repertoire that is not only enriched for hydrophobic CDR-H3s, but for longer CDR-H3s as well. To test the role of long, hydrophobic CDR-H3s in permitting the generation of broadly neutralizing anti-HIV antibodies, Dr. Schroeder will challenge mutant ?D-D[unreadable]FS BALB/c mice, as well as control ?D-DFL and wild-type BALB/c mice, with MPER immunogens obtained from Dr. Haynes. To begin to test the role of tolerance, Dr. Schroeder will also challenge mutant and wild-type DH locus mice on BALB/c, C57BL/6, and C57BL/6 sle1/sle2/sle3 congenic backgrounds, each of which progressively increases susceptibility to the generation of autoreactive antibodies. Dr. George Shaw will evaluate the sera from these mice for the presence of neutralizing antibodies. Drs. Schroeder and Kearney will generate hybridomas from those mice that express neutralizing MPER antibodies and obtain monoclonal antibodies for further study. These anti-MPER monoclonal antibodies will be further screened for neutralization and for autoreactivity. Their binding properties will be examined. Finally, their V domains will be cloned and sequenced to further correlate the relationship between neutralization and CDR-H3 length or hydrophobicity. The creation of vaccines capable of eliciting neutralizing antibodies to HIV has proven more difficult than first expected [7;44]. The HIV envelope spike plays a key role in viral infection, and antibodies directed against the MPER region at the base of the spike are broadly and potently neutralizing. Unfortunately, these antibodies are not only extremely uncommon;they also tend to be autoreactive. The site at which these antibodies bind HIV contains a binding structure that tends to be both larger and enriched for fat soluble amino acids than most such binding structures in other antibodies. We have used genetic techniques to force mice to express antibodies enriched for longer binding structures as well as for longer, more fat soluble binding structures. To test the extent to which these features will enable generation of neutralizing MPER antibodies, we propose to challenge our mutant mice with HIV MPER immunogens, isolate anti-MPER antibodies, test whether these antibodies neutralize HIV, and further correlate structure with function.